Accepted Manuscript

Chitosan and Chitosan Coating Nanoparticles for the Treatment of Brain Dis-
ease

Shuangwen Yu, Xiaolu Xu, Jianfang Feng, Mei Liu, Kaili Hu

PII: S0378-5173(19)30135-8
DOI: https://doi.org/10.1016/j.ijpharm.2019.02.012
Reference: IJP 18150

To appear in: International Journal of Pharmaceutics

Received Date: 30 October 2018

Revised Date: 31 January 2019
Accepted Date: 12 February 2019

Please cite this article as: S. Yu, X. Xu, J. Feng, M. Liu, K. Hu, Chitosan and Chitosan Coating Nanoparticles for
the Treatment of Brain Disease, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/
j.ijpharm.2019.02.012

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Chitosan and Chitosan Coating Nanoparticles for the Treatment of

Brain Disease
Shuangwen Yua,b,1, Xiaolu Xua,b,1, Jianfang Fengc, Mei Liub*, Kaili Hua,d,*
a
Murad Research Center for Modernized Chinese Medicine, Institute of
Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional
Chinese Medicine, Shanghai 201203, China
b
Shanghai Innovation Center of TCM Health Service, Shanghai University of
Traditional Chinese Medicine, Shanghai, 201203, China
c
School of Pharmacy, Guangxi University of Chinese Medicine, Nanning, 530001,
People’s Republic of China
d
Key Laboratory of Smart Drug Delivery, Fudan University, Ministry of Education,
Shanghai 201203, China
*
Corresponding author.
E-mail address: liumeiy@aliyun.com (Mei Liu), kaili-hu@163.com (Kaili Hu)

Abstract

Since the application of chitosan magnetic cationic microspheres in

brain drug delivery by Gallo in 1993, chitosan has been extensively

studied in brain drug delivery. As the only natural alkaline

polysaccharide with good biocompatibility and biodegradability,

chitosan prepared nanoparticles which use chitosan as carrier material

to encapsulate drug have been proved with advantages of protecting

from enzyme degradation, control release and improved bioavailability.

Besides, chitosan can enhance drug permeability across the blood-brain

barrier by affecting the tight junction. And with positive charge on the

1
surface, chitosan nanoparticles can absorb on the negatively charged

cell membrane, thus increase its residence time on the nasal mucosa and

benefit of the delivery of drugs from the nasal cavity to the brain. All

these properties made chitosan nanoparticles especially suitable for

brain delivery. What’s more, the free amino groups on the surface of

chitosan nanoparticles allow specific chemical modification to form a

wide range of chitosan derivatives. Surface modification of the

nanoparticles with tumor targeting peptides like chlorotoxin and

transferrin, can further improve the targeting property to brain tumor.

This article gives a comprehensive review on the advantages and recent

progresses in the treatment of brain disease by chitosan nanoparticles.

Keywords

Chitosan nanoparticles, Drug delivery system, Brain disease,

Blood-brain barrier

1. Introduction

Blood-brain barrier (BBB) is a protective barrier of the central

nervous system (CNS) formed by the tight junctions of endothelial cells,

pericytes, and astrocytes, which allows only small hydrophobic

molecules with molecule weight less than 400 to pass(Betsholtz, 2014;

Bickel et al., 2001; Zhao et al., 2017). Over 98% small molecule drugs
2
and almost 100% large molecule drugs like recombinant protein and

monoclonal antibodies cannot enter the brain. Both the specific

structure and the efflux proteins on the BBB surface limit the entry of

drugs into the brain, so one of the major challenges of brain disease

therapy is to deliver drugs across the BBB to the brain lesion at an

effective concentration(Gao, 2017).

Over the past decades, there were a variety of strategies developed

for improving the concentration of drugs in the brain. Besides direct

strategies like destroy the integrity of BBB by physicochemical method,

there were indirect biological approaches, such as receptor-mediated

transcytosis, which are non-invasive method by modifying ligands on

the drug carrier and specifically targeting receptors at the BBB,

achieving targeted delivery of drugs and reducing the toxic and side

effects of nonselective drug distribution (Gao, 2016; Patel et al., 2010).

Invasive method could deliver drugs into the brain, which present some

risks, such as lack of targeting, traumatic and some of surgical

complications (Fang et al., 2017). It is a promising approach to modify

drug delivery systems with specific targeting molecules and allow

delivery into the brain through specific carrier or receptor on the BBB.

The polymer nanoparticles have gained a great deal of attentions as

a promising CNS drug delivery system for its brain targeting ability and

controlled release property (Fazil et al., 2012). Chitosan is obtained

3
from the N-acetylation of chitin in the presence of hot alkali. It is a

copolymer of N-acetyl-glucosamine and N-glucosamine units linked by

β-(1’4)-glycosidic bonds, and has good biocompatibility,

biodegradability, mucoadhesivity and low toxicity (Fig. 1). The LD50

dose of chitosan is in the same range as sugar or table salt (Kean and

Thanou, 2010). Chitosan has three types of reactive functional groups,

which are amino groups, primary and secondary hydroxyl groups at the

C-2, C-3, and C-6 positions, respectively. These groups allow a variety

of chemical modifications of chitosan that include acylation,

N-phthaloylation, tosylation, reductive alkylation,

O-carboxymethylation, N-carboxyalkylation, silylation, and graft

copolymerization. Lysozyme is the major enzyme that responsible for

the degradation of chitosan in vivo by the hydrolytic action of the

acetylated residue. The degradation rate of chitosan is inversely related

to the degree of crystallinity which is decided by the degree of

deacetylation (Ashrafi and Azadi, 2016; Lanjhiyana et al., 2013;

Saboktakin et al., 2011).

There are a large amount of advantages of chitosan for drug

delivery, which including : 1) the ability of controlling release; 2) free

of toxic organic reagent while manufacturing nanoparticles because

they can dissolve in acidic aqueous solution; 3) a linear polyamine

containing a number of free amine groups that are readily available for
4
crosslinking; 4) cationic chitosan can strongly combined with anionic

material, cell surface and mucous membrane, which benefits to open

tight junction，improve membrane absorption by prolong the residual

time at the site, and so on (Prabaharan, 2008; Wang et al., 2008).

Compared to polyester nanoparticles (PLGA, PLA and PCL

nanoparticles) usually used in brain delivery, chitosan nanoparticles are

unique for their positive charged surfaces and good mucosal adhesion.

These characteristics could help to enhance brain drug delivery, whether

through intranasal or intravenous routes (i.v.). However, chitosan’s poor

water solubility is a major limitation for its application (Babu and

Ramesh, 2017). In addition, the encapsulation efficiency of both

hydrophilic and hydrophobic drugs is poor, which could be overcome

by modifying chitosan or optimizing the preparation method (Babu and

Ramesh, 2017).

Chitosan has been widely modified to meet various biological and

medical needs due to its active functional groups. The common

modification method is chemical modification. The amino groups may

involve in chemical reactions such as alkylation, quaternization,

reacting with aldehydes and ketones, grafting, etc. The hydroxyl

functionality also leads to some reactions like o-acetylation, H-bonding

with polar atoms, cross-linking, grafting, etc(El et al., 2018; Sahariah

and Másson, 2017). Currently, the condensation reaction between amino

5
group and carboxyl group is mainly used to modify the targeted groups

on the surface of chitosan nanoparticles and improve the targeting effect

of nanoparticles. And no obvious toxic effects of modified chitosan

nanoparticles have been reported(Qian et al., 2013; Stephen et al., 2014;

Veiseh et al., 2010).

Actually, the advantages of chitosan made it especially suitable for

nasal drug delivery. Nasal delivery is a method of administration with

the characteristics of non-invasiveness, painless, no aseptic

requirements and simplicity in operation. It is a classical non-invasive

approach direct delivery to the brain and nasal cavity is the only

exposed part of the CNS (Elnaggar et al., 2015; Hanafy et al., 2016; Lin

et al., 2016). One of the major obstacles of nasal delivery is the

scavenging action of nasal cilia, which can clear away the delivered

drug and decrease the absorption time. Low drug permeability of nasal

mucosa is another limiting factor (Patel et al., 2012). However, mucous

adhesion polymers will be less affected through enhancing contact with

the nasal membrane which prolong the absorption time of drugs and

increase the drug concentration gradient. Chitosan has the properties of

biological adhesiveness and can promote drug permeability, which

benefit drug delivery from nasal to brain as a nasal delivery carrier

(Wang et al., 2008). Besides, since chitosan is a positive charged

polymer, it can extend its retention time at the target and increase
6
absorption through the interaction with negative charged cell membrane

(Elnaggar et al., 2015; Mistry et al., 2015; Wang et al., 2008).

Currently, chitosan nanoparticles have been used extensively in

treatment of brain disease including gliomas, Parkinson’s disease,

Alzheimer’s disease, etc (Meng et al., 2018; Shevtsov et al., 2018;

Sridhar et al., 2018; Xu et al., 2017). In this article, the recent

development of chitosan and its derivate nanoparticles in the treatment

of different brain disease were reviewed and future applications are

discussed.

2. Brain glioma

Brain glioma accounts for 80% of all malignant brain tumors, and

has characteristics of fast onset and strong aggressiveness (Abakumov

et al., 2015). It is mainly divided into astrocytoma, dendritic cell tumor,

ependymoma and mixed glioma according to their morphology.

Glioblastoma multiforme is the highest degree glioma of the

astrocytoma.

The current standard treatment for glioma is surgical resection

combined with radiation and chemical therapy. However, it is difficult

to thoroughly remove the tumor edge which infiltrated into the

surrounding normal tissue in the late stage of glioma without

irreversible damage of the patient’s brain function. In addition,

7
high-grade gliomas are highly resistant to radiation therapy, which leads

to about 100% relapse and a median survival of only 12-14 months after

diagnosis (Rizvi et al., 2010). What’s more, the special structure of BBB

to a certain extent limits chemotherapeutics delivery into the tumor and

achieve efficient concentration for treatment (Jose et al., 2016).

2.1 CS nanoparticles via i.v. route

Agrawal et al. (Agrawal et al., 2017) developed a transferrin (Tf)

receptor targeted bioadhesive micelles using D-α-tocopheryl glycol

succinate 1000 (TPGS) conjugated chitosan (TPGS-CS). Antitumor

drug docetaxel (DTX) was loaded through self-assembly. The prepared

micelles have a particle size of 16.12 ± 2.2 nm, a zeta potential of - 1.11

± 0.57 mV and an encapsulation efficiency of 98.9%. The

mucoadhesive chitosan resulted in adsorption-mediated transcytosis,

combined by Tf receptor-mediated endocytosis, leading to better

targeting to Tf receptor overexpressed glioma cells. The in vitro

cytotoxicity results demonstrated that the DTX formulated in the

DTX-TPGS-CS and DTX-Tf-TPGS-CS are 97 and 248 folds more

efficient than free DTX after 24 h treatment. And in vivo

pharmacokinetic results show that the relative bioavailability of

non-targeted drug-loaded micelles and targeted drug-loaded micelles

after 48 h were 2.89-fold and 4.08-fold higher than that of free DTX,

respectively (Fig. 2).

8
2.2 CS nanoparticles via nasal route

Chitosan nanoparticles is a kind of good carrier to deliver

macromolecular therapeutics targeting glioma, especially small

molecular nucleic acids regulating the tumor microenvironment.

Galectin 1 (Gal-1) is a naturally occurring galactose-binding lectin,

which is overexpressed in GBM, and highly associated with tumor

progression (Toussaint et al., 2012). Gal-1 is also a potent immune

suppressor in the tumor microenvironment. It can induce apoptosis of

activated CD8+ T cells, inhibit T cell signaling and block

pro-inflammatory cytokines secretion (Corapi et al., 2018). In order to

specifically inhibit Gal-1 in GBM, Woensel et al. (Van Woensel M et al.,

2016) use chitosan nanoparticle to encapsulate an anti-Gal-1 siRNA at

an encapsulation efficiency of 81 ± 3%. In vitro experiments

demonstrated that the nanoparticles with a diameter of 141 ± 5 nm and

zeta potential of ＋ 32 mV can protect siRNA from ribonuclease

degradation and enhance its delivery from nasal epithelium to CNS by

temporarily by enhance the retention time and penetration on the nasal

mucosa. The delivered siRNA down-regulated the expression of Gal-1

in GBM and inhibited tumor progression in vivo.

Chitosan nanoparticles also draw much interest in loading Chinese

herbal medicines for glioma treatment (Chirio et al., 2014). Yang et al.

(Yang et al., 2015) developed a polyelectrolyte complex nanoparticles

9
(PENPs) by hyaluronic acid and chitosan (HA/CS) for loading

water-insoluble curcuminoid (CUR) to treat glioma. The nanoparticle

shows spherical shape with a diameter of 207 nm and a positive charge

of 25.37 mV. The encapsulation efficiency and drug loading are 89.9%

and 6.5%, respectively. The in vitro release study demonstrated the

sustained release behavior of CUR from CUR-PENPs in 5% glucose

solution after a burst release. In vitro cytotoxicity experiment showed

that blank PENPs in the studied concentrations have no effects on cell

viability of C6 glioma cells, suggesting blank PENPs might be good

biocompatible carriers. Compared to CUR solution, CUP-PENPs

suggested a better dose-dependency in the toxicity on C6 glioma cells.

And the uptake of CUR-PENPs by C6 cells is also higher than that by

CUR solution. Xu et al. (Xu et al., 2017) further synthesized

lactoferrin-coated nanoparticles based on chitosan / hyaluronic acid /

PEG (Lf-Cur-PSNPs) and effectively accumulated in the brain (2.39

times greater than PSNPs).

2.3 CS and derivatives coating

The commonly used chemotherapeutic drugs for glioma in clinic

are temozolomide (TMZ) and carmustine (BCNU), which can cause

alkylating damage of DNA and further inhibit its replications (Qiu et al.,

2014; Rubino et al., 2018). However, O6-methylguanine-DNA

methyltransferase (MGMT), a DNA repair protein, can remove the

10
damage produced by chemotherapeutic drugs. O6-benzylguanine (BG)

has been used to enhance the efficiency of chemotherapeutic drugs

TMZ and BCNU by irreversible prevent the DNA repair as an

inactivator of MGMT. Stephen et al. (Stephen et al., 2014) developed a

magnetic nanoparticle with chitosan-PEG copolymer coating whose

surface is modified through covalent attachment of BG and tumor

targeting peptide CTX (NPCP-BG-CTX). Every nanoparticle contains

150 ± 14 molecular BG and 3 ± 1 molecular CTX, respectively. The

Z-average size of nanoparticle is 76 ± 4 nm and the average zeta

potential is 4 ± 7.4 mV. The release of BG modified on the surface do

not achieve maximum at 24 h under conditions mimicking plasma,

demonstrating a controlled release of BG. CTX has high specificity and

avidity for glioblastoma multiforme (GBM) through recognition of

MMP2 and Annexin A2 on cell surface and facilitates receptor-mediated

transcytosis through BBB endothelial cells (Dardevet et al., 2015). As

the main carrier material, chitosan is not only used to carry BG, but also

beneficial to the surface modification of PEG. Co-treatment with

NPCP-BG-CTX and TMZ via convection-enhanced delivery (CED) can

decrease the BG level in GMB, and enhance cytotoxicity of TMZ in

GBM cells. In vivo antitumor study results showed a 3-fold increase in

median overall survival of NPCP-BG-CTX/TMZ treated in comparison

to NPCP-CTX/TMZ treated animals (Fig. 3). In addition, toxicity

11
evaluations showed there was no difference in liver toxicity between

saline injected and NP injected mice, indicating the innocuous toxic

profile of NPCP-BG-CTX.

The efficiency of BCNU in glioma treatment is limited by its

inability to concentrated in the tumor site. Qian et al. (Qian et al., 2013)

use CS to modify the surface of poly (lactide-co-glycolides)

nanoparticles and constructed PLGA/CS-NPs for targeting glioma.

BCNU was loaded along with BG to improve the efficiency of BCNU.

The core-shell structure contained a PLGA core carried BCNU and CS

coating shell loaded with BG. The diameter of the NPs is around 177

nm and the average zeta potential is 19.6 ± 4.8 mV, with a loading

efficiency of 3.05% and 8.97% for BCNU and BG, respectively. After

intravenous injection, BG in the shell will be released prior to BCNU,

which will reduce the level of MGMT in tumor cell thus increase the

efficacy of the following BCNU. In vitro experiment shows that the

degradation half-life of BCNU-PLGA/CS-NPs encapsulated BCNU

increased 5-fold compared to free BCNU in plasma. Besides, the uptake

of PLGA/CS-NPs by F98 cells is 2.19-fold higher than those of

PLGA-NPs. In vivo antitumor efficacy study also demonstrated that

compared to free BCNU, BCNU-PLGA/CS-NPs have significantly

better antitumor effect. Remarkably, the median survival of

BCNU+BG-PLGA/CS-NPs group is prolonged to 23 days compared to

12
16 days in the BCNU-PLGA/CS-NPs group and 14.5 days in the BCNU

solution group. In addition, safety studies have shown that the carrier

materials have no adverse effects upon the cell growth. Therefore, it is

suggested that CS modification is an effective strategy for loading of a

second chemical to enhance the antitumor effect of clinical

chemotherapeutic drugs in glioma treatment.

TRAIL is a TNF cytokine superfamily member, which forms a

homotrimer that crosslinks death receptors on the cell surface leading to

downstream signaling of apoptosis. TRAIL is an attractive anticancer

agent due to its ability to induce apoptosis in various types of tumors.

Wang et al. developed a targeted iron oxide NP coated with

chitosan–polyethylene glycol–polyethyleneimine copolymer and

chlorotoxin (CTX) for treatment glioblastoma. NP–TRAIL successfully

delivers TRAIL into human T98G GBM cells and induces secretion of

40 pg mL−1 of TRAIL in vitro. Transfected cells show threefold

increased apoptosis as compared to the control DNA bound NPs.

Systemic administration of NP–TRAIL–CTX to mice bearing

T98G-derived flank xenografts results in near-zero tumor growth and

induces apoptosis in tumor tissue (Wang et al., 2015). Veiseh et al.

(Veiseh et al., 2010) developed a nanoparticle by polyethylene glycol

(PEG)-grafted chitosan and polyethylenimine (PEI) to encapsulate a

superparamagnetic iron oxide nanoparticle core. The nanoparticle was

13
further functionalized with siRNA and a tumor-targeting peptide, CTX,

to improve glioma targeting ability. The final nanoparticle has a

diameter of 111.9 ± 52.4 nm, with a zeta potential of＋19.6 ± 5.7 mV,

and an average of 3.8 molecular siRNA and 5 molecular CTX on each

nanoparticle. In this system, PEG-grafted chitosan stabilizes the

nanoparticles from agglomeration. The superparamagnetic core enables

non-invasive real-time monitoring of the nanoparticles by magnetic

resonance imaging. This nanoparticle safely and efficiently delivery the

PEI-binded siRNA in vivo and specifically knockdown the expression

of green fluorescence protein (GFP) in C6/GFP＋glioma cells.

Chitosan nanoparticles can successful carry chemotherapeutic

drugs, siRNAs and natural products to brain (Chen et al., 2017; Chirio

et al., 2014; Danhier et al., 2015; Messaoudi et al., 2014). The chitosan

used for various nanoparticles are different, with different molecular

weights, co-use with other materials like PLGA or use of synthetic

materials such as PEG-CS and TPGS-CS. Nanoparticles are mainly

prepared by ion crosslinking method which generated particles with a

particle size of about 100 nm. However, much smaller micelles can be

prepared by TPGS-CS with solvent casting method, resulting in a

particle size of about 16 nm, and a relatively higher encapsulation

efficiency of 98.9%. Intravenous injection is the main method of

chitosan nanoparticle administration, while intranasal administration is

14
also frequently used. The molecular weight of chitosan, the co-used

material, the preparation method as well as different target

modifications will affect the properties of the prepared chitosan or

chitosan coated nanopartices and thus affecting the effects on brain

disease therapy. In i.v. delivery systems, chitosan is usually used with

other materials to improve the loading, sustained release, cell uptake,

delivery and targeting of drugs. The CS-PEG is a commonly used

chitosan derivative, and modified with PEG can enhance the aqueous

solubility of chitosan and benefits the delivery of water-soluble drugs

such as rivastigmine (RHT), galantamine hydrobromide, and riluzole

etc. Besides, as a linear polyamine containing many cationic amine

groups, chitosan could be used as a carrier to encapsulate proteins,

peptides, and siRNA by mild ion interactions. What's more, cationic

chitosan can strongly combined with anionic cell surface and mucous

membrane, which benefits to open the tight junction and improve

membrane absorption by prolong the residual time (Smith et al., 2004).

The positive electrical property of chitosan nanoparticles can improve

glioma cell uptake, and assist loadings escape from endosome and

delivery into the cytosol after modified with PEI (Yezhelyev et al.,

2008). In relatively low pH environments, such as in tumor tissues,

positive chitosan nanoparticles accumulate preferentially, thus

enhancing the anti-tumor effect (Chung et al., 2010). In addition, there

15
are modifiable free amino groups on the surface of chitosan which can

conjugated to tumor targeting peptide like CTX and Tf, which further

improve the targeting property of chitosan nanoparticles to brain tumor.

The free amino groups can also be methylated or quaternized according

to the nature of the drug and the need of the delivery carrier. After

intranasal administration, apart from the advantages mentioned in i.v.

administration, the positive charge and mucoadhesiveness of chitosan

can also improve the retention time and penetration of chitosan

nanoparticles into the nasal mucosa. The CS and derivatives coating can

exert all kinds of properties of chitosan depend on different design.

Moreover, the free amino groups can also bind to probes such as

fluorescein to observe the distribution of nanoparticles in the body.

Chitosan is a promising excipient in pharmaceutics because of its good

biodegradation and biocompatibility. Using chitosan as nanoparticle

materials provides a good carrier for chemotherapeutic drugs that of

poor glioma bioavailability and stability.

3. Alzheimer’s disease (AD)

The increasing prevalence rate of neurodegenerative disorder has

been a seriously public health problem. AD was first proposed by Alois

Alzheimer in 1906, is characterized by the extracellular deposition of

amyloid-β (plaques), the intracellular aggregation of

16
hyperphosphorylated tau protein (tangles), and a progressive

degeneration of intra-cortical projecting neurons (Dyrba et al., 2018).

Clinical manifestations of AD include cognitive decline and progressive

behavioral disorder, among which first appeared is the lack of

short-time memory. Along with the progression of the disease, some

other impairment of cognitive function gradually revealed, such as the

continued deterioration of intellectual and social skills, memory loss,

personality changes and inability to take care of oneself (Muntimadugu

et al., 2016).

The pathogenesis of AD that causes neuronal degeneration and

cognitive impairment is not yet clear. The current strategy for treating

symptoms of AD is to improve cognitive function and memory for a

better quality of life. The cholinergic hypothesis of AD pathogenesis

suggested that cholinergic neurons and acetylcholine (ACh) synthesis

are highly affected in AD patients leading to memory impairment and

cognition defectiveness. And inhibiting acetylcholinesterase (AChE)

using cholinesterase inhibitors (Hanafy et al., 2016) shows good effect

on increasing ACh concentration in brain of AD patients (Ouyang et al.,

2017) .

3.1 CS nanoparticles via i.v. route

The currently FDA-approved cholinesterase inhibitors include

donepezil, RHT, galantamine, and tacrine (Contestabile, 2011). Tacrine

17
is the first ACh inhibitor approved for the treatment of AD (Reichman,

2003). However, due to the first pass effect, the absolute bioavailability

of tacrine is only about 17% ± 13%. To solve this problem, Wilson et al.

(Wilson et al., 2010) prepared tacrine-loaded chitosan nanoparticles to

sustained release tacrine. The prepared nanoparticle with a diameter of

41 ± 7 nm, a zeta potential of 34.7 ± 1.5 mV and a loading efficient of

10.86 ± 0.30%. Following intravenous injection, the chitosan

nanoparticles significantly increased the half-life of tacrine and

improved the efficiency of brain delivery.

3.2 CS nanoparticles via nasal route

RHT is a good candidate for AD therapy as an inhibitor of both

acetyl-cholinestrase (AChE) and butrylcholinestrase enzyme. It is 4–17

times more specific for inhibiting AChE in brain as compared to AChEs

in heart and blood. While its hydrophilicity limits its oral delivery and

restricts its entry into the brain. It always requires frequently

administration which leading to severe cholinergic side effects.

Chitosan is a good carrier material which can increase the delivery of

encapsulated chemicals from nose to brain (Sarvaiya and Agrawal,

2015). Fazil et al. (Fazil et al., 2012) developed RHT-chitosan

nanoparticles (CS-RHT-NPs) to increase RHT’s brain delivery after

intranasal administration. The prepared nanoparticle with particle size

of 185.4 ± 8.4 nm, zeta potential of 38.4 ± 2.85 mV and a loading

18
capacity of 43.37 ± 3.9% showed sustained release property over 24 h.

The brain targeting index of RHT by RHT (intravenous), RHT

(intranasal), and CS-RHT-NPs (intranasal) were 0.235, 0.790 and 1.712

respectively at 30 min, which proved direct nose to brain transport

bypassing the BBB by intranasal administrated CS-RHT-NPs. Besides,

the brain concentration of RHT was significantly higher after intranasal

administrated CS-RHT-NPs than those after intravenous or intranasal

administration of RHT solution.

In another research, Hanafy et al. (Hanafy et al., 2016) used

chitosan nanoparticles to deliver galantamine hydrobromide via the

intranasal route for the treatment of AD. The results indicated that the

complexation of chitosan with galantamine hydrobromide significantly

reduced the level and activity of AChE protein in the rat brain compared

to oral and nasal delivery of the galantamine hydrobromide solution. In

addition, the galantamine hydrobromide-loaded chitosan nanoparticles

showed no sign of toxicity which demonstrated its good

biocompatibility (Wahba et al., 2016).

Piperine (PIP) has been reported as a phytopharmaceutical with

neuroprotective potential in AD. Elnaggar et al. (Elnaggar et al., 2015)

developed PIP-chitosan nanoparticles (PIP-NPs) for AD treatment. The

particle size, zeta potential and encapsulation efficiency of the prepared

nanoparticles are 248.50 nm, +56.30 mV and 43.37 ± 3.9%. In vitro

19
drug release study showed that the percentage of PIP release from

PIP-NPs is only 10% compared to 82% of free PIP after 2 h in PBS

indicating a sustained-release of PIP from nanoparticles.

Neuroinflammation is a prominent feature of AD and other chronic

neurodegenerative diseases. This study revealed for the first time the

mechanism of PIP in treating AD via anti-inflammatory and

anti-apoptosis effects. A rat memory impairment model was established

by injecting colchicine into the lateral ventricle of Wistar rats and

PIP-NPs and donepezil was administrated for 13-14 days by intranasal

administration. The results show that PIP-NPs significantly improved

the escape latency from the second day of treatment compared with the

negative control and donepezil group. In retention memory, compared

with the positive control, both PIP-NPs and donepezil group

significantly prolong the retention time, indicating an improved

cognitive function. Besides, PIP-NPs also protects PIP from enzymatic

degradation and reduces its allergy. In addition, brain toxicity study

reports no neurotoxic effect to any of the studied formulations.

3.3 CS and derivatives coating

Trimethyl chitosan (TMC), a quaternization derivative of chitosan,

is a water-soluble, non-toxic material that carries positive charge under

physiological conditions. As a cationic polymer, TMC can promote the

active transport of nanoparticles by adsorption-mediated transcytosis.

20
Wang et al. (Wang et al., 2010) prepared TMC surface-modified poly (D,

L-lactide-co-glycolide) (PLGA) nanoparticles (TMC/PLGA–NP) to

load coenzyme Q10(Co-Q10). The results suggested a higher

accumulation of TMC/PLGA-NP in the cortex, paracoele, the third

ventricle and choroidplexue epithelium after i.v. injection (Fig. 4).

Behavioral tests results showed that Co-Q10-TMC/PLGA-NPs

significantly improved memory impairment compared with

Co-Q10-PLGA-NPs. Water maze test performed on transgenic AD

model mice shows that Co-Q10-TMC/PLGA-NP significantly improved

the spatial memory ability of mice compared to PLGA-NP and free

Co-Q10. Co-Q10-TMC/PLGA-NP decreased senile plaques in brain and

normalized MDA, GSH-Px and CAT levels also confirmed the good

brain target ability of TMC/PLGA-NP. Moreover, cytotoxicity

evaluation results showed that TMC/PLGA–NP is a promising drug

carrier without any significant cytotoxic effects.

4. Parkinson’s disease (PD)

PD is the second most common neurodegenerative disease which

characterized by striatal dopamine depletion due to denaturation of

dopaminergic neurons in the substantia nigra (Dorsey et al., 2007; Kalia

and Lang, 2015). The major symptoms of PD are loss of mobility,

resting tremor, bradykinesia, rigidity and postural instability. The

21
pathogenesis of PD is not yet clear, perhaps are the result of multiple

factors such as aging, mitochondrial dysfunction, oxidative stress and

apoptosis (Beilina and Cookson, 2016; Van Den Eeden SK et al., 2003).

Sustained oxidative stress will cause failure of the antioxidant defense

mechanism and produce free radical oxygen. The existence of persistent

oxidative stress in the substantia nigra pars compacta of PD patients

may be one of its important pathogenesis.

4.1 CS nanoparticles via nasal route

Levodopa is the most effective drug in the treatment of PD in

clinic. However, the clinical response to levodopa is variable and

unreliable due to its unstable oral absorption and fluctuated plasma

concentration. Bromocriptine (BRC), an ergot derivative with dopamine

receptor agonist activity, is widely used clinically to delay and minimize

unwanted motor fluctuations due to long-term use of dopamine in the

treatment of PD. Md et al. (Md et al., 2014, 2013) investigated the

effect of intranasally administrated chitosan nanoparticles on

brain-targeting efficiency of BRC. The BRC loaded chitosan

nanoparticles (CS-NPs) prepared by ionic gelation method has a particle

size of 161.3 ± 4.7 nm, a zeta potential of +40.3 ± 2.7 mV, and a loading

capacity of 37.8 ± 1.8%. After oral administration of BRC, only a small

amount can reach the target site due to incomplete absorption and

extensive metabolism. The striatum-plasma ratio of BRC was lower

22
than the pituitary-plasma ratio, indicating that the drug was limited

outside the brain tissue by the BBB. The prepared BRC-loaded chitosan

nanoparticles can carry the drug across the nasal mucosal and to protect

the drug from degradation in the nasal cavity. Furthermore, the mucosa

adhesion property of chitosan can prolong its residence in the nasal

cavity to improve absorption of the nasal mucosa by delaying

mucociliary clearance. Pharmacodynamics results showed that the

model group’s maximum cataleptic behavior is 281.7 ± 9.1 s after 120

min, while the value is 152.7 ± 17.5 s in the BRC solution treated group,

and 47.7 ± 8.6 s in the BRC loaded CS-NPs group. The BRC solution

and BRC loaded CS-NPs-treated groups were shown to have improved

systemic stiffness and exercise capacity, especially in mice treated with

BRC loaded CS-NPs (Md et al., 2014).

4.2 CS and derivatives coating

Apart from chemotherapeutics, neurotrophic factors such as glial

cell-derived neurotrophic factor (GDNF) can also be used to provide

neuroprotection to dopaminergic neurons. However, the application of

GDNF has been limited in clinical due to its short half-life and rapid

degradation after in vivo administration, in addition to difficulties in

crossing the BBB caused by its inherent hydrophilicity, large molecular

weight and the charge (Gao et al., 2013; Hernando et al., 2018).

Gartziandia et al. (Gartziandia et al., 2016) studied the in vivo

23
neuroprotection effect of GDNF encapsulated in chitosan coated lipid

carrier (CS-NLC-GDNF) in a 6-OHDA partially lesioned rat model

after intranasal administration. The nanoparticle, with a particle size of

about 130 nm and encapsulation efficiency of 98%, showed protective

effects on PC-12 cells against 6-OHDA toxin in vitro and on

amphetamine-induced rotational behavioral in vivo. After 7 weeks

continuous supply of CS-NLC-GDNF, there was a significantly

decrease in the number of rotations per minute and the number was

reduced by 80% until the end of the study. What’s more, the density of

tyrosine hydroxylase fibers in the striatum and substantia nigra were

significantly improved in the CS-NLC-GDNF group, indicating a better

protective effect than that of free GDNF.

5. Spinocerebellar ataxia (SCA)

SCA is a neurodegenerative disease characterized by loss of

cerebellum and spinal cord cells, which affects motor coordination,

body posture and balance (Ashizawa et al., 2018). One of the hallmarks

of in vitro SCA models is the over-expression of ataxin protein. As

chitosan is frequently used as a non-viral nanocarrier materials to

delivery siRNA to the target gene, Malhotra et al. (Malhotra et al., 2013)

synthesized TAT-tagged (PEG)ylated chitosan polymer to encapsulate a

functional siRNA against the Ataxin-1 gene (CS-PEG-TAT-NPs) for the

24
treatment of SCA. A SCA model was constructed in vitro by transfected

the neuronal cells with liposomes that contained the Ataxin-1 plasmid.

The western blot analysis results showed that ataxin-1 protein

expression of Neuro2a cells was totally inhibited after 48 hours,

indicating that nanoparticles could successfully achieved 100% siRNA

transfection in neuronal cells. Cytotoxicity studies demonstrated that the

modified CS-PEG-TAT nanoparticles did not induce any significant

toxicity when compared to

the untreated cells.

Neurodegenerative diseases are CNS diseases, in which the

successful delivery of drugs to the brain region is critical to the

treatment (Gao, 2016). Therefore, many therapeutic strategies tend to

use chitosan nanoparticles to enhance brain targeted delivery of CNS

therapies. The preparation of chitosan nanoparticles for the treatment of

neurodegenerative disease is mainly based on ion-crosslinking method.

Besides, nanoparticles are mainly prepared by chitosan alone, only a

few co-used with other materials. Unmodified chitosan nanoparticles

usually administrated by intranasal route, which is mainly attributed to

the fact that the positive chitosan nanoparticle can effectively penetrate

the nasal mucosa and directly enter the brain region. Compared with

modified chitosan materials and chitosan combined with other materials,

it is more convenient to use chitosan as the carrier nanoparticles alone

25
by intranasal delivery. Intranasal delivery is an ideal way for

administration of chitosan nanoparticles due to its inherent

mucoadhesive characteristics. In an experimental study of the

relationship between drug delivery methods and efficacy, researchers

found that the efficacy of nasal delivery of chitosan nanoparticles was

higher than that of intravenous delivery (Fazil et al., 2012; Md et al.,

2014). Pathways of intranasal delivery to brain are shorter than those of

the intravenous delivery because it can directly enter the brain along the

olfactory nerve or trigeminal nerve thus bypass the BBB (Agrawal et al.,

2018).

6. Cerebral ischemia

Cerebral ischemia is a brain disease caused by temporary or

permanent decrease in cerebral arterial blood flow. The clinical

character of cerebral ischemia is partial neurological deficit which is

one of the most leading causes of disability and mortality (Nazam et al.,

2008). Cerebral ischemia will eventually lead to the death of neurons,

especially in caudate-putamen and frontal sensorimotor cortices, and

induce various sensorimotor and motor deficits such as lack of

coordination, dyskinesia and occasional partial paralysis.

6.1 CS nanoparticles via i.v. route

It has been reported that the inhibition of caspase-3 enzyme can

26
increase the survival of neuronal cells after cerebral ischemia. The

peptide Z-DEVD-FMK is a specific caspase-3 inhibitor while it cannot

pass the BBB and diffuse into the brain tissue. Therefore, it is necessary

to develop an efficient delivery system to provide effective drug

concentrations in the brain. Aktas et al. (Aktaş et al., 2005) designed

chitosan nanospheres conjugated with PEG bearing anti-caspase peptide

Z-DEVD-FMK, with a particle size of 149.73 ± 1.85 nm, zeta potential

of +16.06 ± 3.43 mV，and an encapsulation efficiency of 31.13 ± 1.61%.

Chitosan nanospheres were further modified with monoclonal antibody

OX26 using the avidin (SA)-biotin (BIO) technique. After intravenous

administration, most of the fluorescently labeled CS-PEG-OX26 NPs

were localized in the brain and outside of the intravascular compartment.

Electron microscopy of brain tissue confirmed that this new targeted

drug delivery system successfully transferred Z-DEVD-FMK to brain

tissue following intravenous administration.

6.2 CS nanoparticles via nasal route

Ahmad et al. (Ahmad et al., 2016) prepared rutin-encapsulated

chitosan nanoparticles (RUT-CS-NPs) via ionic gelation method for the

treatment of cerebral ischemia. The particle size of RUT-CS-NPs is

92.28±2.96 nm with a PDI of 0.206 ± 0.006. The zeta potential is 31.04

± 1.91 mV, and drug loading and encapsulation efficiency is 39.48 ±

3.16% and 84.98 ± 4.18%, respectively. The results of ex-vivo

27
permeation studies on nasal mucosa showed that the permeability of

RUT-CS-NP was greater than 80% after 24 h, while that of rutin

solution was only 19.01%. The interaction between the positive charged

chitosan of RUT-CS-NPs and the negative charged mucosal membrane

greatly enhanced the drug permeability compared to free RUT. And the

adhesiveness of nanoparticles prolongs its residence time in the nasal

cavity and increases its penetration and continuing delivery to the brain

by temporarily open tight junctions of the BBB. The

UPLC-ESI-Q-TOF-MS/MS detection results showed the uptake of

RUT-CS-NPs into the brain was three times higher than that of the free

RUT, and the relative bioavailability of RUT-CS-NPs was significantly

increased compared with that of the free RUT. This also results in a

significantly reduced volume of cerebral infarction of the RUT-CS-NPs

group compared with the cerebral ischemia model group (Fig. 5).

Adenylate cyclase A1 receptor agonist (CPA), a derivative of

adenosine, is a potent and selective agonist of adenosine Al receptor and

inhibits the release of aspartic acid and glutamic acid in rat cortical

areas reduced by ischemia, eventually leading to neuroprotective action.

Dalpiaz et al. (Dalpiaz et al., 2008) designed CPA loaded chitosan

nanoparticles. In vivo study in rats showed there was no CPA found in

rat cerebrospinal fluid and brain sections after intravenous

administration of free CPA. In contrast, after CPA-loaded chitosan

28
nanoparticles i.n. administration, CPA was detected in the cerebrospinal

fluid and the concentration increased from nM to µM, two orders of

magnitude higher than those obtained at systemic level. The

concentration of CPA in the olfactory is about 0.1 ng/mg, indicating

that the application of chitosan as a carrier can successfully deliver

anti-ischemic drugs into the CNS after intranasal administration.

6.3 CS nanoparticles via intraperitoneal (i.p.) route

Riluzole has been used as a potent neuroprotective agent to treat a

number of diseases such as PD, multiple sclerosis, cerebral ischemia

and so on. It is the only available drug for the treatment of motor neuron

disease. However, its application has been limited because of the poor

aqueous solubility, short half-life and side effects such as lung toxicity

at high concentration (Borderías-Clau et al., 2006). Verma et al. (Verma

et al., 2016) synthesized chitosan conjugated N-isopropyl acrylamide

(NIPAAM) nanoparticles coated with tween 80 to load riluzole (NR).

The particle size of NR is around 50 nm. Drug-loaded nanoparticles

were given intraperitoneally 1 h after middle cerebral artery occlusion,

and rats were dissected for assessment of various parameters after a

reperfusion period of 24 h. Triphenyl tetrazolium chloride staining

showed that the infarct size treated with NR was significantly decreased

compared to model groups, even at a low concentration. The occlusion

of the middle cerebral artery simultaneously triggers some

29
inflammatory molecules, such as NOS, NF-kB and COX-2, which

eventually lead to cell death. However, the immunohistochemistry

results showed that NOS-2, NF-kB and COX-2 expression were

significantly reduced compared with the model group after NR

treatment. These results indicated that nanoparticles had the ability to

carry riluzole across the BBB and exert prominent neuroprotective

effects even at very low drug concentration. By greatly reducing the

required concentration, the NR also decreased the side effects of

riluzole.

Growth factors like basic fibroblast growth factor (bFGF) suppress

cell death by acting at several points on death pathways and,

additionally, promote regeneration, which makes it a promising agent

for treatment of stroke. Yemisci et al.(Yemisci et al., 2015) showed

that that after i.p. administration bFGF-loaded chitosan nanoparticles

modified with PEG and TfRMAb significantly decreased the infarct

volume in cerebral ischemia mice.

Cerebral ischemia, a reason for stroke, generally causes permanent

deterioration of the CNS. In order to improve the effect of treatment, it

is necessary to increase medicine concentration in the brain. Hence,

drug delivery system has been developed for some therapeutic

chemicals, such as riluzole, rutin, CPA and Z-DEVD-FMK. The

molecular weights of chitosan were 150-370 kDa, but all had higher
30
deacetylation degree. However, because of the difference in the drug

encapsulated, preparation methods and material properties, the particle

size is quite different. When prepared by ionic crosslinking, the particle

size of Riluzole-NP is about 50 nm, while the Z-DEVD-FMK-CS NP

modified with PEG and SA/OX26 is 636 nm. When prepared by

spray-drying, the particle size of CPA-CS was as high as 20 µm.

Despite the difference in size, they all benefit on the cationic surface of

chitosan nanoparticle which effectively penetrates the BBB or directly

enter the brain region.

7. Conclusion

In this review, recent development of chitosan drug delivery systems

for treatment of brain disease has been highlighted. Due to the presence

of the BBB, researchers have been directing their efforts to increase

drug concentrations in the brain region. Chitosan nanoparticles have

achieved considerable maturity in treating brain diseases in the past 20

years. Many kinds of chitosan nanoparticles have been proved to

improve the therapeutic efficacy of different brain diseases due to its

biocompatibility, biodegradability, low toxicity, controlling release,

mucoadhesivity, modifiability and effective uptake by nasal mucosal

cell and tumor cells. Chitosan nanoparticles are prepared in a

hydrophilic environment, thus the amount of loaded drug is generally

31
lower for lipophilic molecules than for hydrophilic molecules (Grenha,

2012; Luppi et al., 2010). What’s interesting, chitosan nanoparticles are

also often used to deliver therapeutics from natural products in addition

to chemical and macromolecular therapeutics, successfully increased

the brain concentration of them which are originally difficult to

penetrate through BBB after i.n. administration. This may because

intranasal administration is commonly used for brain delivery of natural

products and chitosan nanoparticles could enhance the nasal residence

time and uptake by nasal mucosal due to the positively charged surface.

In recent years, more and more attention is paid on natural products

which have good effects in the treatment of brain disease like tumor and

neurodegenerative diseases with low toxicity. However, most of the

active compounds are water-soluble and are difficult to infiltrate into the

brain through BBB. Therefore, a carrier with good biocompatibility like

chitosan is favorable, which not only protects the stability of the drug,

but also effectively improves the concentration of the drug in the brain.

Overall, chitosan-based nanoparticles are excellent carriers for the

treatment of brain diseases.

Funding

This work was supported by National Natural Science Foundation of

China [81202924, 81773909, 81703711]; Shanghai Rising-Star

32
Program of China [13QA1403400]; Shanghai talent development funds

[201665]; Excellent Medical Profession Scholarship of Shanghai

municipal commission of health and family planning [2017YQ060] and

Fudan University & The Open Project Program of Key Lab of Smart

Drug Delivery, Fudan University [SDD2017-01], Ministry of Education,

China.

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49
Dise Co-us Chito Preparat Modifi Siz Zeta EE (%) Adminis Outcome
ase ed san ion cation e pote tration
carrier mole method (n ntial method
materi cular m) (mV
al weig )
ht

Tab.1 Chitosan-based nanoparticles for treatment of brain glioma

50
 Iron 5 kDa Co-prec PEG, 76 4± 50 CED Threefold
oxide ipitate CTX ±4 7.4 molecu increase in
core lar BG median
/ overall
nanopa survival of
rticle NPCP-BG-C
TX/TMZ
treated in
comparison
with the
control group
PLGA 50 Emulsio - 17 19.6 3.05 i.v. Targeted
kDa n 7 ± 4.8 for delivery to
solvent BCNU glioma,
evaporat and reversed
ion 8.97 drug
for BG resistance,
and
improved
anti-tumor
Brai
efficacy
n
- 50 Ionic - 14 32 81± 3 i.n. Down-regula
glio
kDa gelation 1± ted the
ma
5 protein level
and
expression of
Gal-1 in
GBM
Iron - Co-prec PEI, 11 19.6 3.8 - Enhanced
oxide ipitate PEG, 1.9 ± 5.7 molecu internalizatio
core CTX ± lar n and
52. siRNA knockdown
4 / of targeted
nanopa gene
rticle expression in
tumor cells

Iron - Co-prec PEI, 57. 18.6 - i.v. Threefold

oxide ipitate PEG, 1± ± 1.3 increased
core CTX 1.4 apoptosis of
glioblastoma
cell in vitro
compared

51
 with control
group, and
resulted in
near-zero
tumor
growth and
induced
apoptosis in
tumor tissue
TPGS - Solvent Tf 16. 1.1 ± 98.9 i.v. 248 folds
casting 12 0.6 effective than
method ± free DTX in
2.2 C6 glioma
cells, and
improved the
relative
bioavailabilit
y by
4.08-fold
compare
with free
DTX
Hyalu 50 - - 20 25.4 89.9 - Increased
ronic kDa 7 uptake and
acid killing effect
on C6 tumor
cells

Dis Co- Chito Preparatio Modifi Size Zeta EE D Admini Outcome

ease used san n method cation (nm) pote (% L stration
carri mole ntial ) C method
er cular (mV (%
mat weig ) )
erial ht
- - Spontaneo Polyso 41 ± 7 34.7 - 10. i.v. Sustain-rel
us rbate ± 9 ease,
emulsifica 80 1.5 ± improved
AD
tion 0.3 the
efficiency
of brain

52
 targeting
- 750 Ionic - 185.4 38.4 85. 43. i.n. Improved
kDa gelation ± 8.4 ± 3 37 nasal
2.9 ± ± delivery
3.5 3.9 efficiency
- 200 Ionic - - - - - i.n. Reduced
kDa gelation the level
and
activity of
acetylcholi
nesterase
protein
- 100- Ionic - 248.5 56.3 81. 43. i.n. Improved
300 gelation 0 0 70 37 cognitive
kDa ± functions
3.9 and
cognitive
ability
PL 100 nano- - 146.7 21.0 8.8 - i.v. Improved
GA kDa precipitati ± 5.1 ± memory
/ on method 2.9 impairmen
10k t and
Da enhanced
the spatial
memory
ability,
decreased
senile
plaques in
brain and
normalized
MDA,
GSH-Px
and CAT
levels
- Medi Ionic 161.3 40.3 84. 37. i.n. Reduced
um gelation ± 4.7 ± 2 8 catalepsy
mole 2.7 ± ± and
cular 3.5 1.8 akinesia
PD weig
ht
NL - melt-emul 136.7 30.0 98. i.n. Significant
C sification ±14.1 10 ly
technique ± improved
53
 0.3 the density
6 of tyrosine
hydroxylas
e (TH+)
fibers in
the
striatum
and
substantia
nigra
- Low Ionic PEG, 50-10 - - - - Successfull
mole gelation TAT 0 y and
cular efficiently
weig transfected
ht functional
siRNA
SC
causing
A
suppressio
n of ataxin
protein in
neuronal
cells in
vitro
Tab.2 Chitosan-based nanoparticles for treatment of neurodegenerative disease

Dise Co-use Chito Preparat Modific Size Zeta E D Administ Outcom

ase d san ion ation (nm) poten E LC ration e
carrier molec method tial ( (% method
materia ular (mV) % )
l weigh )

54
 t
N-isopr - - Tween8 50 - - - i.v. Decreas
opyl 0 ed
acryla infarct
mide size and
the
expressi
on of
NOS-2,
NF-kB
and
COX-2
- Medi Ionic - 92.3 31.0 84 39. i.n. Reduce
um gelation ± ± 1.9 .9 5 d the
molec 2.9 ± ± volume
ular 4. 3.2 of
weigh 2 cerebral
t infarctio
n
- 160 Spray-d - 196 - - - i.v.; i.p. Increase
kDa rying 0± d the
50 concentr
CI
ation of
CPA in
the CSF
- < 150 Ionic PEG, 149. 16.1 - 31. i.v. Facilitat
kDa gelation OX26 7± ± 3.4 1 ed
1.8 ± nanopar
1.6 ticle
transloc
ation
into the
brain
tissue
- Ionic PEG, 747 - - - i.v. Promote
gelation TfRMA ±42 d
b nanopar
ticles
across
the BBB
and
reduced
the
infarct
55
 volume
Tab.3 Chitosan-based nanoparticles for treatment of cerebral ischemia

Graphical abstract

56